Journal: Biochemical pharmacology

Article Title: mTORC2 is an important target for simvastatin-associated toxicity in C2C12 cells and mouse skeletal muscle - Roles of Rap1 geranylgeranylation and mitochondrial dysfunction.

doi: 10.1016/j.bcp.2021.114750

Figure Lengend Snippet: Fig. 7. Simvastatin increases mitochondrial ROS production, which can be mitigated by antioxidants and which contributes to impaired function of mTORC2 and cytotoxicity. C2C12 myoblasts or myotubes were treated for 24 h with 10 μM simvastatin and/or 10 to 20 μM MitoTEMPO and/or 10 to 50 µM geranylgeraniol. MitoSOX Red dye was used to quantify mitochondrial superoxide production. DMSO 0.1% was used as a negative control and 100 μM antimycin A as a positive control (1 h exposure) A. Mitochondrial O2 •- accumulation in C2C12 myoblasts transfected with control siRNA or Rap1 siRNA. B. Mitochondrial O2 •- accumulation in C2C12 myotubes treated with simvastatin or antimycin A. C. Membrane integrity (AK release) of C2C12 myotubes treated with simvastatin, MitoTEMPO, or anti mycin A. D. Intracellular ATP of C2C12 myotubes treated with simvastatin, MitoTEMPO or antimycin A. E. mTORC2 activity assessed by phosphorylation levels of Akt (Ser473) in C2C12 myotubes treated with simvastatin, MitoTEMPO or antimycin A. F. Quantification of Akt (Ser473) phosphorylation shown in E. Data represent the mean ± SEM of at least three independent experiments. *P < 0.05 versus the respective DMSO control; +P < 0.05 versus 10 μM simvastatin or 100 μM antimycin A. SMV: simvastatin, GGOH: geranylgeraniol, MITOT: MitoTEMPO, ANT.A: antimycin A.

Article Snippet: Then, membranes were incubated overnight with the following primary antibodies diluted 1:1000 in the blocking solution: phospho-Akt (Ser473), Akt (4060 and 2920; Cell Signaling Technology, USA), phospho-S6 Ribosomal protein (Ser235/236), S6 ribosomal protein (4858 and 2217; Cell Signaling Technology, USA), Rheb (13879; Cell Signaling Technology, USA), Rictor (2114; Cell Signaling Technology, USA), Ras (610001; BD Biosciences), unprenylated Rap1 and total Rap1 (sc-398755 and sc-373968 respectively; Santa Cruz Biotechnologies, USA).

Techniques: Negative Control, Positive Control, Transfection, Control, Membrane, Activity Assay, Phospho-proteomics

Journal: Biochemical pharmacology

Article Title: mTORC2 is an important target for simvastatin-associated toxicity in C2C12 cells and mouse skeletal muscle - Roles of Rap1 geranylgeranylation and mitochondrial dysfunction.

doi: 10.1016/j.bcp.2021.114750

Figure Lengend Snippet: Fig. 8. Effects of simvastatin on mTORC1 and mTORC2. Simvastatin impairs the function of mTORC2 by inhibition of Rap1 and by mitochondrial damage. Impaired function of mTORC2 reduces the function of Akt (impaired phosphorylation of Ser473), which limits the function of mTORC1 and increases the expression of atrogin- 1. Impaired function of mTORC1 reduces the activity of S6rp. Cell death associated with simvastatin can be explained by apoptosis (via inhibition of Akt and mitochondrial damage) and necrosis (mitochondrial damage).

Article Snippet: Then, membranes were incubated overnight with the following primary antibodies diluted 1:1000 in the blocking solution: phospho-Akt (Ser473), Akt (4060 and 2920; Cell Signaling Technology, USA), phospho-S6 Ribosomal protein (Ser235/236), S6 ribosomal protein (4858 and 2217; Cell Signaling Technology, USA), Rheb (13879; Cell Signaling Technology, USA), Rictor (2114; Cell Signaling Technology, USA), Ras (610001; BD Biosciences), unprenylated Rap1 and total Rap1 (sc-398755 and sc-373968 respectively; Santa Cruz Biotechnologies, USA).

Techniques: Inhibition, Phospho-proteomics, Expressing, Activity Assay